Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.     

Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.     

Amidation of C3 at the thiolester site: stimulation of chemiluminescence and phagocytosis by a new inflammatory mediator

We studied the ability of particle-bound and fluid-phase C3b monomers and monomeric amidated C3 (prepared by treatment of purified human C3 with ammonium chloride or methylamine) to stimulate chemiluminescence of human polymorphonuclear leukocytes (PMN) and monocytes and to promote phagocytosis in the absence of antibody. Particle-bound C3b evoked chemiluminescence from both PMN and monocytes, and fluid-phase C3b (0.5 mg/ml) elicited significant chemiluminescence from PMN but not from monocytes. Amidated forms of C3, both particle bound and fluid phase, were potent stimulators of chemiluminescence from phagocytic cells and caused a significantly greater response than did C3b. The phagocytosis of 1-micron microspheres by PMN and monocytes was enhanced by coating them with purified C3b in an antibody-free system. Microspheres coated with amidated C3 were avidly phagocytized, and to a greater degree than were C3b-coated microspheres. In a direct binding assay with tritiated monomeric C3b and amidated C3, the affinity of the PMN complement receptor type 1 for C3b (Ka = 4.9 X 10(7) L/M) was similar to that for amidated C3 (Ka = 5.7 X 10(7) L/M). However, there was a fourfold increase in the number of apparent binding sites for amidated C3. This increase did not reflect binding of amidated C3 to the complement receptor type 3, because blocking of CR3 with the monoclonal antibody OKM 10 failed to decrease binding of amidated C3. In sites of increased ammoniagenesis, such as the kidney in chronic renal failure, amidated C3 may play a role as an inflammatory mediator by stimulating oxidative metabolism in phagocytic cells.     

Structure and possible ureide degrading function of the ubiquitous urease of soybean

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.

The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection (“Western Blot”). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.

The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

     

Purification and infectivity ofEntomophaga grylli (Fresenius) Batko Pathotype 2 againstMelanoplus differentialis (Thomas) [Ort.: Locustidae]

The life stages ofEntomophaga grylli (Fresenius) Batko Pathotype 2 were purified and separated by centrifugation in Percoll^R density-gradient medium. The ranges of buoyant densities for germinated resting spores, germ conidia, and resting spores respectively were: 1.040–1.050, 1.055–1.085, and 1.080–1.120 g/ml. Cuticular invasion by germinated germ conidia was the means by whichMelanoplus grasshoppers became infected. Scanning electron micrographs revealed germination of germ conidia on the visible host integument at 100% RH, but not at 90% RH. Significantly higher mortality (P<0.05) was obtained after 3 weeks with grasshoppers incubated in constant light than in constant dark for 24 h following treatment. The disease was not transmitted by ingestion of any life stage. Contribution N^o 85-153-J, Department of Entomology. Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506.     

Peptidoglycans as promoters of slow-wave sleep. II. Somnogenic and pyrogenic activities of some naturally occurring muramyl peptides; correlations with mass spectrometric structure determination

The structures of components of the sleep-promoting material purified from human urine were established by fast atom bombardment-mass spectrometry, as reported in the accompanying paper (Martin, S. A., Karnovsky, M. L., Krueger, J. M., Pappenheimer, J. R., and Biemann, K. (1984) J. Biol. Chem. 259, 12652-12658). We report here that two substances isolated from that preparation, viz. N-acetylglucosaminyl-1,6 -anhydro-N-acetylmuramyl-Ala-gamma-Glu-diaminopimelyl-Ala) and that compound lacking the terminal alanine, are active as somnogens. Cerebro-intraventricular administration of 1 pmol of the glycotetrapeptide was sufficient to induce prolonged excess sleep in rabbits. A similar substance obtained from Brevibacterium divaricatum in which the free carboxyls of the glutamic and diaminopimelic moieties, indicated above, were amidated (N-acetylglucosaminyl-1,6-anhydro-N-anhydromura-myl-Ala-iso- Gln- epsilon-amido-diaminopimelyl -Ala-Ala) was not active as a promoter of slow-wave sleep. Deamidation of this peptide to a mixture of the free dicarboxylic forms produced a somnogenic substance. Our findings show that in addition to the muramyl form of peptidoglycan monomers, the anhydro muramyl form, with no reducing end, is compatible with somnogenic activity. Furthermore, the data obtained with a natural product amplify our earlier observations with smaller synthetic molecules of the importance of amidation/deamidation in the structure-activity relationships of muramyl peptides.     

On the binding of histone H1 in chromatin

Nucleosomal subunits isolated from rabbit thymus nuclei in 0.04 M K₂SO₄-0.02 M Tris, pH 7.4 were devoid of histone H1, while whole chromatin prepared in the same buffer contained the full complement of histone H1. The question is asked why histone H1 dissociates from the subunits but not from the high molecular weight material. We propose that, at physiological salt concentrations, histone H1 is not bound to linker DNA as depicted in the current models; rather, alternate attachment sites, present only in the polymer, are involved.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.